In an enzymatic reaction, usually, it is preferred to carry out the reaction in a homogeneous system wherein an enzyme is dissolved. On the other hand, after the reaction is completed, it is desired that the enzyme can be readily separated from the reaction system. In view of this, immobilized enzymes having reversible soluble-insoluble properties have been studied. As such immobilized enzymes, for example, the following immobilized enzymes have been known: (1) lysozyme immobilized on alginic acid (see Biotechnology and Bioengineering, 16, 1553-1556. (1974)); (2) trypsin immobilized on an acrolein-acrylic acid copolymer (see Biotechnology and Bioengineering, 18, 587 (1976)); (3) penicillin amidase, alcohol dehydrogenase and the like immobilized on a mixture of a polymethacrylic acid and poly-4-vinyl-N-ethylpyridine (see Biotechnology and Bioengineering, 24, 237, (1982)); (4) enolase, pyruvate kinase, peroxidase and the like immobilized on a casein polymer (see Agricultural and Biological Chemistry, 48, 2435-2440 (1984)); and (5) glucose isomerase, glucoamylase and the like immobilized on a polyacrylamide derivative (see Japanese Patent Publication No. 40474/1983).
However, these known techniques have various disadvantages. For example, the immobilized enzyme (1) is liable to be inactivated because it is required to reduce pH to 2.5 to precipitate it. In addition, since the enzyme is insoluble in an organic solvent, it can not be used when a substrate is soluble in an organic solvent or a solution containing an organic solvent but insoluble in water. In the immobilized enzymes of (2) and (5), pH should be changed to a great extent in order to attain a complete precipitated state as well as a completely dissolved state and, therefore, pH suitable for the reaction is limited. The immobilized enzyme (3) requires strict conditions for immobilization and is liable to be inactivated. In addition, the use thereof is limited because the enzyme is readily precipitated in the presence of a monovalent cation such as sodium or potassium ion which usually exists in an enzymatic reaction and, therefore, such an ion can not be added. Moreover, the immobilized enzyme (4) has some problems. For example, a carrier thereof is difficult to prepare and the enzyme is liable to leak. Further, it is difficult to use in an organic solvent system since the enzyme is insoluble in an organic solvent. Thus, development of an immobilized enzyme having reversible solubility (i.e. soluble-insoluble immobilized enzymes) which are free from the above disadvantages has been desired.
As the result of intensive study, the present inventors have succeeded in preparing an immobilized enzyme which can readily and reversibly transfer from a dissolved state to a precipitated state and have a high activity as well as a high solubility even in an organic solvent or in a solution containing an organic solvent.